WO2011121097A2

WO2011121097A2 - Method and device for analysing molecular bonds

Info

Publication number: WO2011121097A2
Application number: PCT/EP2011/055062
Authority: WO
Inventors: Michael Geisler; Thorsten Hugel; Roland Netz
Original assignee: Technische Universität München
Priority date: 2010-03-31
Filing date: 2011-03-31
Publication date: 2011-10-06
Also published as: DE102010014657A1; WO2011121097A3

Abstract

A scanning force microscope (1) is used for a measurement process in order to analyse the molecular bond to a probe molecule (5) of individual molecules which are in a solution. To this end, the bond parameters can be determined for the adsorbed state of the probe molecule (5), wherein the probe molecule (5) is adsorbed on an adsorption surface (7), or for a desorbed state of the probe molecule (5).

Description

description

Method and device for studying molecule ^bindings

The invention concerns methods for the investigation of moles ^¬ külbindungen, wherein:

a measuring head and an adsorption body with an adsorption surface are provided;

- is the distance between the measuring head which is provided with a Sondenmo ^¬ lekül, and the adsorption body, to the adsorbing the probe molecule is at least partially adsorbed changed;

- Using a force measuring device, the force is measured, which is mediated by the probe molecule between the measuring head and adsorbent body;

a force-displacement curve is determined which shows the dependence of the measured force on the change in the distance between the measuring head and the adsorption body;

- By evaluation of force plateau of the force-displacement curve, a desorption size of the probe molecule at the adsorption in quasi-steady state equilibrium is determined.

The invention further relates to an apparatus for carrying out the method.

Such a method and device are known from WO 2008/081044 AI. The adhesion of single molecules to a substrate surface is investigated by the known method. For this purpose, the individual molecules are bound to a carrier polymer. The probe molecule formed in this way is attached to a measuring head whose distance to the substrate surface is variable. A force measuring device is used to measure the force imparted by the probe molecule between the measuring head and the substrate surface. On the basis of the measured values, a force-displacement curve can be determined, which determines the dependence of the measured force of the change in the distance between the measuring head and Adsorptionskörper shows. The plateau levels of the power plateaus can be determined with great accuracy. In this respect, it is possible that the Desorptionskraft Sondenmole- CRWT to determine on the adsorption in the quasi-stationary Equilibrium ^¬ weight. Since the Desorptionskraft between the probe molecule and the substrate surface is mediated by the Einzelmole ^¬ cules, can investigate the adhesion of the single molecules to the substrate surface with the known method.

The presence of dissolved compared to the Sondenmole ^¬ kül small molecules such as organic compounds or chemical process residues are often undesirable for gesundheitli- chen or quality-technical reasons. One

However, detection is difficult due to the poor reactivity of many ^substances . Further solvent additives and Kosolute (eg ions, ligands, enzymes, other small Biomo ^¬ leküle, including small proteins), protein folding, the assembly of polymers, the adhesion or the peeling of a macromolecule Doomed of a boundary layer ^¬ things. At the same time, the interaction of cosolutes with a macromolecule is influenced by their proximity to an interface. But the characterization of Bindungsverhal- least smaller molecules from solution to a macromolecule is of great importance for many technical applications, which take place in solution: for example the production of molecular markers, medical diagnostics, example ^¬, the diagnosis of blood sugar in case of diabetes Mellitus, the functionality of molecular Sensortechnolo ^¬ gies, such as "Lab on a chip" applications, and

Enzyme- or DNA-based field-effect transistors for biomedical engineering. The specific ligand adsorption at isolated, well-defined binding sites can be determined by classical methods such as fluorescence [1, 2] or radioactive labeling. cation [3]. In contrast, only a method for determining the number of bound ligand for ternary systems is for the unspezifi ^¬ specific binding, such as blood glucose, available. Such ternary systems consist of macromolecule, solvents and cosolutes. A strategy for the complete characterization within the technically important four-component system, which additionally contains an extensive surface, does not yet exist.

The binding characteristics in ternary systems can calorimetry [4], densitometry [5] or Gleichgewichtssedi ^¬ mentation [6] are determined. The most commonly used method is isothermal titration calorimetry (= ITC).

However, this requires a certain minimum amount of molecules to provide reliable information about changes in free energy and binding constants. The measurement accuracy increases with the amount of macromolecules used. In addition, self-assembly and aggregation limit the applicability of the ITC to hydrophilic and amphiphilic ones

Molecules [4].

Alternative methods for determining the molecular-ligand interaction in thermodynamic equilibrium in the vicinity of a surface are by their spatial and temporal

Resolution limited. Very specific binding can, as already mentioned, be investigated by fluorescence. For this purpose, however, a very dilute solution of both macromolecules and Kosoluten is necessary, which often represents a great simplification compared to the technically relevant system. In addition, a chemical modification of the Kosolute is necessary. Another common method for determining physical binding parameters makes use of radioactive contrast agents. Here, the spatial resolution is determined by the range of the beta-emitter, which is far above molecular dimensions. Similar problems learn with the resolution powers are given for ellipsometry, X-ray and neutron scattering,

In ZHANG, H.Y. and MARKO J.F., Maxwell's Relationship for Single-DNA Experiments: Monitoring Protein Binding and Double-helix Torque with Force-extension Measurements, Physical Review E 77 (3), 2008 will review the use of force curves in the

Extensions of double-stranded DNA (= dsDNA) are generated, theoretically analyzed to characterize the binding between dsDNA and other molecules. The results obtained require the peculiarities of deletions of dsDNAs and are therefore not suitable for studying the binding of single molecules to any macromolecules. Based on this prior art, the object of the invention is therefore to specify a method with which the binding of small individual molecules to any macromolecules, in particular single-stranded DNAs, can also be investigated.

This object is achieved by a method and an apparatus having the features of the independent claims. In dependent claims advantageous embodiments and developments are given.

In the method, probe molecules are repeatedly withdrawn from the adsorbent surface of the adsorbent body. In this case, the activity of the individual molecules is varied in a region adjacent to the adsorption surface of the adsorption body and a desorption size is determined as a function of the activity of the individual molecules. In addition, by

Determination of the relationship between desorption size and activity, binding parameters can be determined which describe the binding of the individual molecules to the probe molecule in the adsorbed and desorbed state of the probe molecule. In this way it is possible to make the binding behavior smaller To study molecules on macromolecules in the adsorbed and desorbed state.

It should be noted that the term activity in this context is to be understood as a generalization of the concentration and thus also includes concentrations.

In a particular embodiment of the method, the desorption size is determined on at least one further adsorption surface of at least one further adsorption body as a function of the activity of the individual molecules. The material properties of the different adsorption surfaces should differ. On the basis of the relationships determined between desorption size and activity for the various adsorption surfaces, binding parameters can then be determined which describe the binding of the individual molecules to the probe molecule in the desorbed state and to the states of the probe molecule adsorbed on the various adsorption surfaces. With this particular embodiment of the method, the accuracy with which the binding parameters can be determined can be increased considerably.

In a further embodiment of the method, the desorption size is determined at different temperatures as a function of the activity of the individual molecules. Based on between desorption size and activity determined

Contexts, the binding parameters can then be determined, which describe the binding of the single molecules to the Sondenmole- kül in the desorbed and adsorbed states of the Son ^¬ denmoleküls at the various temperatures. The additional data can also increase the accuracy in determining the binding parameters. In a further embodiment, the desorption force is determined in the evaluation of the power plateau. By determining the relationship between desorption power and activity telt, will bond parameters that are determined that describe the binding of single molecules to the probe molecule in adsorbing ^¬ th and desorbed state of the probe molecule. In this way it is possible to study the binding behavior of small molecules to polymers in the adsorbed and desorbed state.

The binding parameters which can be determined by the method are in particular the binding constants for the binding of the

Single molecules to the probe molecule in the equilibrium state and the mean binding site length, which is equal to the length of the probe molecule divided by the number of binding ^{Stel ¬} len. The binding constants and the binding site lengths can each be determined for the adsorbed state of the probe molecule and for the desorbed state of the probe molecule.

In this case, it can be assumed that the Bindungskon ^¬ stante and the mean length of binding sites in the desorbed state is independent of the nature of the Adsorptionsflä ^¬ surface. For the determination of the relationship between desorption power and activity, the binding parameters concerning the desorbed state of the probe molecule can therefore be equated in each case.

The relationship between Desorptionskraft and activity can be determined by adjusting an equalizing function of measured values of the Desorptionskraft, wherein the measured values are plotted against the change in the activity and the parameters of the equalizing function, the binding constants and the mittle ^¬ re binding sites length for the adsorbed and desorbed state of the probe molecule , In particular onskraft in matching different compensation functions on measurements of desorption, which have been used in the investigation of different adsorption areas that Genauig ^¬ ness can be substantially increased in the determination of binding parameters. In a further embodiment, the desorption length is determined in the evaluation of the power plateau. From the dependence of the desorption length on the activity of the

Single molecules can then be determined a number of the individual molecules bound to the probe molecule.

In order to be able to ren variie- the activity of the individual molecules in the region adjoining the Adsorpti ^¬ onsfläche of the adsorbent region, the individual molecules are dissolved in a solvent. In this case, a gaseous or liquid solvent can be used.

Further advantages and features of the invention will become apparent from the following description, are explained in the embodiments of the invention with reference to the drawings in detail. Show it:

Figure 1 is an atomic force microscope or short force microscope (= Atomic Force Microscope = AFM);

FIG. 2 shows a representation of the measuring head of the force microscope from FIG. 1 in the adsorbed (A) and desorbed (B) state of a probe molecule attached to the measuring head;

Figure 3 is a diagram with force-displacement curves having a force plateau;

FIG. 4 shows a diagram with measured detachment lengths;

Figure 5 is a diagram with measured adhesive forces;

Figure 6 is a representation showing the interaction between

Probe molecule, single molecules and adsorption ^¬ surface illustrated in the adsorbed state; Figure 7 is a diagram showing the interaction between

Probe molecule and single molecules in the desorbed state illustrated;

Figure 8 is a diagram showing the relationship between

Activity and concentration for a solution shows Glukoselö ^¬;

Figure 9 is a diagram with measured adhesive forces at

various adsorption surfaces confining ^¬ Lich the force curves determined;

FIG. 10 shows a diagram with the number of bound single molecules per probe molecule length in the desorbed and adsorbed state for different adsorption surfaces;

Figure 11 is a diagram in which the desorption in

Dependence on activity at different temperatures is shown; and

Figure 12 is a chart showing the number of bound Einzelmo ^¬ leküle per probe molecule length in the adsorbed and desorbed state at various tempera ^¬ ren.

1 shows an atomic force microscope or just microscope force micro ^¬ (= Atomic Force Microscope = AFM) which has a measuring ^¬ head 2 which is mounted on a leaf spring. 3 The leaf spring 3 is fixedly connected to a holder 4 at an end opposite the measuring head 2. The measuring head 2 may be provided with a probe molecule 5, which is typically a macromolecule, in particular a

Polymer acts.

The force microscope 1 further has an adsorption ^¬ body 6, the one the measuring head 2 and the probe molecule. 5 having facing adsorption 7. The Adsorptionskör ^¬ per 6 is located in a container 8, which is filled with a Lö ^¬ sungsmittel 9. The container 8 is on a

Sample holder 10 is arranged, which can be moved so that the distance between the measuring head 2 and the adsorption ^¬ surface 7 is varied.

It should be noted that the solvent may in principle be both gaseous and liquid.

The force microscope 1 is further equipped with a laser 11 ^{ausgestat ¬} Tet, the light beam 12 to one of the adsorption

6 facing away from the rear side 13 of the leaf spring 3 sends. Of the

Light beam 12 is directed from there to a photodetector 14 with a sectored photodiode. That from the photodetector

14 supplied measuring signal is using an evaluation unit

15 evaluated, which may be, for example, a commercially available computer. The evaluation unit 15 is also used to control the sample holder 10.

With the force microscope 1, the desorption force can be measured, with which the probe molecule 5 at the adsorption

7 is held. For this purpose, the sample holder 8 is actuated such that the distance between the measuring head 2 and the adsorption surface 7 increases. Since the Sondenmole ^¬ kül 5 adheres to the adsorptive surface 7, the measuring head 2 is moved slightly in the direction of the adsorption surface 7 down, which leads to a deflection of the light beam 12th This Auslen ^¬ effect can be detected using the detector 14th Since the spring constant of the leaf spring 3 is known, it can be concluded that the desorption force.

FIG. 2 shows the leaf spring 3 of the force microscope 1 above the adsorption surface 7. The measuring head 2 is respectively provided with the probe molecule 5, which is shown in the adsorbed state (A) and in the desorbed state (B). FIG. 3 shows a diagram with a superimposition of force-displacement curves 16, which has force plateaus 17 and a plateau stage 18. The force-displacement curves were recorded in an experiment described in more detail below, in which a poly (allylamine) molecule (PAAm) was used as the probe molecule 5 and an oxidized diamond surface as Adsorptionsoberflä ^¬ surface 7. FIG. 3 shows the superimposition of 20 typical force-displacement curves 16 recorded with one and the same PAAm molecule under equilibrium conditions, as shown by the velocity-independent force plateaus 17.

By adjusting fit line 19 to the motor 17, a plateau Desorptionskraft _of F can be determined, which is equal to the force required to pull the probe molecule 5 while maintaining a quasi-steady state of equilibrium of the adsorption. 7 Also of interest is also a desorption length L which is equal to the length of the adsorbed on the adsorption 7 portion of the special molecule 5. The product of L and Desorptionslänge Desorptionskraft F _of which corresponds to a surface 20 is equal to the free desorption.

FIGS. 4 and 5 show typical measured value distributions for the desorption length L and the desorption force F _des . The measured value distributions of the desorption length L and the desorptive force F _des include the results of 100 approximation and separation cycles of the leaf spring 3 in investigating the adsorption behavior of a PAAm molecule in glucose solution on an oxidized diamond surface.

In the following, some thermodynamic relations are derived, with the aid of which various binding parameters can be determined from the measurement results shown in FIGS. 3 to 5. In Figure 6, the fully adsorbed to the adsorption surface 7 probe molecule 5 is shown in the presence of Einzelmo ^¬ lekülen 21 which are dissolved in the solvent. 9 The single molecules 21 attach themselves to binding sites 22 of the adsorbed probe molecule 5 and detach again from the binding sites 22. The attachment process is described by the rate constant K _a ^on and the detachment process by the rate constant K _a ^off . FIG. 7 shows the probe molecule 5 in a further state in which the probe molecule 5 is desorbed from the adsorption surface 7. The individual molecules 21 attach themselves to the binding make ^¬ 22 of the desorbed probe molecule 5 and 22 from the binding sites The Anlagerungsvor- dissolve again transition is described by the rate constant K _d ^on and the release operation by the rate constant K _d ^off.

The rate of attachment dG _{a / d} / dt in the adsorbed and desorbed state of the probe molecule is proportional to the concentration c of the single molecules 21 and the number of free binding sites 22 (N = N _ges x [1 - Θ]) along the probe molecule 5. B ^ L _{= K} ".cN _8a - (l-0 _a , _d ) (1)

The removal rate d9 _{a / d} / dt is proportional to the proportion of occupied binding sites (Ng _es x Q _a / _d ):

The dynamic equilibrium is reached when the rate of addition of new single molecules 21 is equal to the rate of detachment of already attached single molecules 21. By equating the accumulation and

Removal rates and dissolution after Θ are obtained with the rate constant (K _a / _d = _a / _d ⁰ⁿ / _a / ci ⁰ ) each the Langmuir isotherm:

C ald (3)

The occupancy rate Θ thus increases with increasing concentration c of the individual molecules 21. This relation applies only to ideal mixtures. For non-ideal mixtures, the

Concentration c by the activity a of the single molecules 21 to replace. For the activity a and the concentration c the relation holds: α = γ - c (4) where γ is the activity coefficient. The following applies: a = exp (/ kT) (5) where k is the Boltzmann constant and T is the temperature. The effect of the desorption force on the probe molecule has not yet been considered. The behavior of the system formed by probe molecule 5 and annealed single molecules 21 can now be described using a thermodynamic potential similar to the large-canon potential for open systems. This potential results from the state sum Z of the system. For the differential of this potential the following applies: d (kT In Z) _ald = L dF _of + Ν _αΙά άμ + T _ald dL (6) where L is the desorption _{length of} the probe molecule 5, F is _the desorption force acting on the probe molecule 5, N is the number the occupied binding sites 22 along the probe molecule ^¬ molecule 5, μ the chemical potential of the single molecules 21 and τ a line voltage of the single molecules 21 along the probe molecule is 5, which is equal to the free energy per unit length ^¬ unit. In the equilibrium state, equation (6) follows: d ^T a / d N Θ a, ld (7) άμ L d ald where d is the distance between the total binding sites along the probe molecule 5. The distance d is hereinafter also referred to as binding site length d. By combining Equation (7) with Equation (3), the change in line voltage τ results in a change in the activity of the single molecules 21: kT

d * a "/ d

During the desorption of the probe molecule 5 are out of the adsorption ^¬ surface 7, the adsorbed on the adsorption portions 7 of the probe 5 and the desorbed portions each in thermal equilibrium. The equilibrium at the portion of the probe molecule 5, which at the

Adsorption surface 7 is adsorbed, is described by the binding ^¬ constant K _a and the binding site length d _a . In contrast, the state of the portion of the probe molecule 5 desorbed from the adsorption surface 7 is determined by a binding constant K _d and a binding site length d _d . The change in the line voltage Δτ between Adsor ^¬ biertem and desorbed state of the probe 5 is therefore equal to: kT kT

AT = -ln (1 + aK _a ) ln (1 + aK _d ) (9) d "d ,,

This difference in the line stress τ mainly contributes to the fact that the desorption force depends on the activity of the single molecules 21: F (a) = F (a = 0) + Ar (10)

It should be noted that, based on the calculated binding constants K, the binding energy of a single binding site can also be calculated:

AG = -RTIn K (11) The procedure described in more detail below by means of a specific experiment makes it possible to detect small single molecules 21 in solution and to characterize their binding to the macromolecule or polymer used as probe molecule 5 both in the dissolved and in the surface adsorbed Status. The influence of the solved

Single molecules 21 on the interaction between probe molecule 5 and adsorption surface 7 due to the different binding behavior of the single molecules 21 to the probe molecule 5 can be used to detect the presence of the individual molecules 21 in the solvent 23.

By the translation of bond equilibria into measurable desorption forces, it is also possible to determine binding parameters such as the binding constants K, bond site lengths d, binding energies and the number of bound single molecules 21 per length. These binding parameters are determined not only for the state in solution of the probe molecule 5, but also for the case in which the probe molecule 5 adheres to the adsorption surface 7.

The procedure was tested using poly (allylamine) in contact with 316L surgical stainless steel or with oxidized diamond. By evaluating the obtained force-displacement curves 16 and applying the basic thermodynamic formulas given in equations (1) to (9)

Relations can be closed to the above binding parameters. The probe molecule 5 used is a single poly (allylamine) molecule (PAAm) which is covalently bonded to the tip of the measuring head 2 of the leaf spring 3 (AFM cantilever). The covalent attachment ensures the necessary long-term stability ^¬ [7] with, and to measure the same probe molecule 5 for the period from several hours to different adsorption faces 7 in glucose solutions of different concentrations, the Desorptionskräfte. In the on zelmolekülen 21 it is in particular Glukosemole ^¬ molecules. The concentration in this case was varied between 0 and 1 M. The tip with the probe molecule 5 was brought into contact with the adsorbing surface 7 for one second to be adsorbed thereon. The subsequent desorption of the probe molecule 5 at a rate of 1 μιη / s was carried out under equilibrium conditions, as was apparent from the force plateau 17 [8].

The probe molecule 5 from FIGS. 6 and 7 is thus a PAAm molecule adsorbed on the adsorption surface 7 in FIG. 6 and stretched in solution in FIG. The single molecules 21 are further gebil ^¬ det of D-glucose molecules. The binding constants K _a = K _a ^on / K _a ^off for the addition of glucose molecules to the PAAm molecule 5 in the adsorbed state on the adsorption surface 7 and the Bindungskonstan ^¬ th K _d = K _d ^on / K _d ^off for the PAAm molecule in solution as well as the number of glucose molecules bound in the respective state can now be determined on the basis of the desorption force as a function of

Be determined glucose activity.

It is again pointed out that precise Bestim ^¬ mung of binding parameters by the Gibbs Adsorptionsiso ^¬ thermenregion presupposes in non-ideal solutions, the measurement of the force as a function of glucose type in place of the Glukosekonzentra- tion. In the experiments described here, therefore, the values for the mole fraction x for Glucose in activities a converted according to a = γχ and thereby the activity coefficient of [9] and [10] used.

FIG. 8 shows the relationship between glucose concentration and glucose activity. From Figure 8 is ersicht ^¬ Lich, that the properties of the solution are similar to those of an ideal solution for average concentrations weakly interacting glucose molecules.

In particular, at a concentration with a mole fraction below 0.05, the relationship between concentration and activity can be approximated by a linearly increasing curve 23. In this range, the activity coefficient is constant and close to 1. Therefore, there is only a small error when using concentrations instead of activities. However, there may be systems that make the use of activities essential for accurate determination of binding parameters.

FIG. 9 shows the measured values for the desorption power of PAAm at various glucose concentrations on stainless steel (open triangles) and on oxidized diamond (filled triangles) at 31 ° C. The Desorptionskraft on oxi ^¬ diertem diamond increases with the concentration of glucose, while the values point to a stainless steel non-monotonic course. The illustrated with solid lines power boost ^¬ ven 24 and 25 illustrate the result of a common off ^¬ same bill. The force cures 24 and 25, the graphical representation of the in equation (9) are reproduced function Δτ with those intended for the respective adsorption surface 7 bonding parameter K _{a /} d and d _{a / d} , which under the

Assuming that the values K _d and d _d for the desorbed state are the same in each case.

Importantly, the compensation calculation is performed on both sets of data obtained with different materials. As a result, the accuracy of the method can be significantly increased. The values obtained for binding parameters, including the length of a gluco ^¬ sebindestelle d and the corresponding equilibrium constant K, therefore, are extremely reliable for the two states. FIG. 10 shows the number of occupied binding sites 22 per unit length. A curve 26 shows the number of accumulated glucose molecules in the desorbed state, another curve 27 the number of accumulated glucose molecules adsorbed to the diamond surface and a third curve 28 the number of accumulated glucose molecules adsorbed to the steel surface. The curves 26 to 28 arising in accordance with equation (7) from the derivative of the force curves obtained for the respective items to ^¬ by the compensating calculation. For concentrations above about 0.2 M, the number of added glucose molecules exceeds the PAAm molecule near the steel surface ^¬ (curve 28) is the one in solution (curve 26). This results in the non-monotonous course of the force curve 25. With the method described here, the small differences can be detected by varying the

Temperature caused.

Figure 11 shows the temperature dependence of glucose binding to the adsorbed and dissolved PAAm molecule. FIG. 11 shows in particular the measured values for the desorption power of PAAm on oxidized diamond in glucose solutions of different concentration at 31 ° C. (open triangles) and 46 ° C. (filled triangles). The force curves 29 and 30 are the result of matching calculations with which the functions according sliding ^¬ chung (9) have been adapted to the plotted measured values.

In turn Figure 12 shows the number of bound Glukosemo ^¬ leküle depending on the activity. Curve 31 relates to the desorbed state at 46 ° C., curve 32 to the adsorbed state at 46 ° C., curve 33 to the desorbed state at 31 ° C., and curve 34 to the adsorbed state at 31 ° C. The force curves 29 and 30 can also be determined to increase the accuracy together with the force curves 24 and 25 in a common compensation calculation.

Table 1 shows the results of the adjustment calculation. A look at Table 1 shows that for the diamond substrate _, the adsorption state binding constant K _{a is} about 8% greater than the binding constant K _d for the desorbed state at 31 ° C and even about 20% greater at 46 ° C. Thus, more glucose molecules are bound to the PAAm molecule in its surface-adherent state. The additionally bound glucose molecules dissolve during

Desorb the PAAm polymer, resulting in the measured

Increase in desorption force leads. Also, the distance between two adjacent binding sites increases during the transition from the adsorbed to the desorbed state.

For the stainless steel surface of the course of the force curve can be explained 25 by the superposition of different glucose binding affinities ^¬. Up to a concentration of about 0.2 M, corresponding to an activity a of about 5 × 10 ^-3 , more glucose binds to the dissolved state in ^comparison to the state of the PAAm molecule adsorbed on the surface. Consequently, the number of bounds increases

Glucose molecules in forced desorption of the PAAm molecule by the additional binding of glucose molecules from the surrounding glucose solution. However, at higher concentrations, more glucose binds to the surface adsorbed PAAm molecule compared to its extended state in solution. Consequently, glucose molecules must go into solution, and the PAAm molecule loses bound glucose molecules while the PAAm molecule is stripped from the metal surface. This is most likely due to the different affinity of glucose for the substrate material itself. At low concentrations, the sugar is rejected from the metal surface and shows a reduced tendency to bind to the PAAm molecule when the PAAm molecule is at the surface. As the amount of glucose on the adsorption layer 7 increases, so does the number of adsorbed glucose until a dense layer of glucose molecules has formed on the adsorption layer 7. Now the Bin ^¬ making behavior is similar to that on the oxidized diamond surface.

Instead of the desorption force, the change in the desorption length L as a function of the concentration or activity of the individual molecules 21 can also be measured. Due to the attachment or detachment of single molecules 21 to the probe molecule 5, there are changes in the desorption length. By measuring them as a function of activity or concentration, the exact number of bound molecules can be determined as long as the change in length per single molecule is known.

Finally, it should be noted that features and properties associated with a particular

Embodiment described can also be combined with another embodiment, except if this is excluded for compatibility reasons. Finally it is noted that the plural ^¬ closes in correspond to the requirements ^¬ chen and in the description, the singular, unless otherwise provided by the context. In particular, when the indefinite article is used, it means both the singular and the plural. Table 1:

Binding parameter of D-glucose to PAAm on stainless steel 3161 and oxidized diamond at different temperature.

Stainless steel 316L O-diamond O-diamond

Temperature 31 ° C 31 ° C 46 ° C

K _d 1300 1300 1400

d _d []] 2.40 2.40 2.68

AG _d [kJ mol ^"1 ] 18.13 18.13 18.32

K _a 114 1404 1789

d _a [Ä] 0.99 2.15 2.55

AG _a [kJ mol ^"1 ] 11.98 18.33 18.94

Literature:

[1] M. Gavutis, S. Lata, J. Piehler, Nature Protocols 2006, 1, 2091

[2] S. D'Auria, A. Ausiii, A. Marabotti, A. Varriale, V. Scognomiglio, M. Staiano, E. Bertoli, M. Rossi, F. Tanfani, Journal of Biochemistry 2006, 139, 213th

[3] M.S. Yang, H.C.M. Yau, H.L. Chan, Langmuir 1998, 14, 6121.

[4] A. Velazquez-Campoy, E. Freire, Nature Protocols 2006, 1, 186.

[5] S.N. Tinesheff, Proceedings of the National Academy of Sciences of the United States of America 2002, 99, 9721.

[6] C. Ebel, H. Eisenberg, R. Ghirlando, Biophysical Journal 2000, 78, 385.

[7] M. Geisler, T. Pirzer, C. Ackerschott, S. Lud, J. Garrido,

T. Scheibel, T. Hill, Langmuir 2008, 24, 1350.

[8] M. Geisler, T.Hugel, Advanced Materials 2010, 22, 398-402.

[9] Y.F. Hu, Z.C. Wang, Journal of Chemical Thermodynamics 1997, 29, 879.

[10] K. Miyajima, M. Sawada, M. Nakagaki, Bulletin of the Chemical Society of Japan 1983, 56, 1620.

Claims

claims

1. A method of studying molecular bonds, in which:

a measuring head (2) and an adsorption body (6) with an adsorption surface (7) are provided;

- the distance between the measuring head (2), with a

Probe molecule (5) is provided, and the adsorption body

(4), on whose adsorption surface (7) the probe molecule (5) is at least partially adsorbed, is changed;

- Using a force measuring device (11-14) the force is measured, which is mediated by the probe molecule (5) between Mess ^¬ head (2) and adsorbent body (6);

a force-displacement curve (16) is determined which shows the dependence of the measured force on the change in the distance between the measuring head (2) and the adsorption body (6);

- by evaluating force plateau (17) of the force-displacement curve (16) has a Desorptionsgröße (F _of, L) of the probe molecule

(5) is determined on the adsorption surface (7) in quasi-stationary equilibrium,

d a d u r c h e s e n c i n e s, d a s s

an activity of individual molecules (21) is varied in a region adjoining the adsorption surface (7) of the adsorption body (6),

- the Desorptionsgröße (F _of, L) is determined depending on the activity of the individual molecules (21), and that

- Binding parameters are determined on the basis of a determined between desorption size (F _des , L) and Akti ^¬ vity (24, 25, 29, 30) binding parameters, the binding of the single molecules (21) to the probe molecule (5) in the adsorbed and desorbiert Describe the state of the probe molecule (5).

2. The method according to claim 1,

characterized in that the desorption size (F _des , L) at least one further

Adsorption surface (7) of at least one further adsorption body (6) as a function of the activity of the individual moons. leküle (21) is determined, and that based on between De- sorptionsgröße (F _of, L) and activity determined together ^¬ hanging (24, 25) to determine the binding parameters, the binding of the individual molecules (21) to the probe molecule (5) in the desorbed state and in the different ones

Describe adsorption surfaces (7) adsorbed states of the probe molecule (5).

3. The method according to claim 1 or 2,

characterized in that the desorption size (F _des , L) at different temperatures depending on the activity of the individual molecules (21) is determined and determined on the basis of determined between desorption size (F _des , L) and activity relationships (24, 25) the binding parameters describe the binding of the single molecules (21) to the probe molecule (5) in the desorbed and adsorbed states of the probe molecule (5) at the different temperatures.

4. The method according to any one of claims 1 to 3,

characterized in that in the evaluation of the power plateau (17) the desorpti- onskraft (F _des ) is determined.

5. The method according to claim 4,

characterized in that as the binding parameter to be determined the binding constants (K _s , K _b ) for the binding of the single molecules (21) to the

Probe (5) in the equilibrium state and the average binding site length (d _s , d _b ) are determined, the

Binding constants (K _s , K _b ) and the binding site length (d _s , d _b ) respectively for the adsorbed on the respective adsorbent surface (7) and those of the respective adsorption surface (7) desorbed state of the probe molecule (5) are determined.

6. The method according to claim 5,

characterized in that for the determination of the binding parameters (K _b , d _b ), those binding parameters which are assigned to the state of the probe molecule (5) desorbed by the adsorption surfaces (7) are set equal.

7. The method according to any one of claims 4 to 6

d a d u r c h g e k e n c e s, the binding constant is a binding energy (AG) per s

Single molecule (21) is determined.

8. The method according to any one of claims 4 to 7

characterized in that the binding parameters are determined by adjusting a compensation function (24, 25) on measured values of the desorption force (F _deS ), the measured values being plotted against the change in activity and the parameters of the compensation function being the binding constants (K _s , K _b ) and the mean binding sites ^¬ length (d _s , d _b ) for the adsorbed and desorbed state of the probe molecule (5).

9. The method according to any one of claims 1 to 3

In the evaluation of the force plateaus (17), the desorption length (L) is determined and from the dependence of the desorption length (L) on the activity of the single molecules (21) a

Number of bound to the probe molecule (5) single molecules (21) is determined.

10. The method according to any one of claims 1 to 9

The individual molecules (21) can be dissolved in a solvent (9).

11. The method according to claim 10

characterized in that a gaseous or liquid solvent (9) is used.

12. Device for studying molecular bonds with:

an adsorption body (6) provided with an adsorption ^surface (7),

- A measuring head (2) whose distance from the Absorptionsflä ^¬ che (7) is variable,

- A force measuring device (11-14), with a force acting between the measuring head (2) and adsorption surface (7) is Meßbai, and with

an evaluation unit (15) connected to the force measuring device (11-14),

The evaluation unit (15) is set up to carry out a method according to one of claims 1 to 11.